CN103656672B - Hyaluronic acid-adriamycin bonding medicine and preparation method thereof - Google Patents
Hyaluronic acid-adriamycin bonding medicine and preparation method thereof Download PDFInfo
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Abstract
The invention provides a kind of hyaluronic acid-adriamycin bonding medicine, have structure shown in formula (I) or formula (II), wherein, n is the hyaluronic degree of polymerization, 1≤n≤1290.The present invention with hyaluronic acid, sodium cyanoborohydride and amycin for raw material, prepare hyaluronic acid-adriamycin bonding medicine, because raw material hyaluronic acid has good biological activity and biocompatibility, the hyaluronic acid-amycin therefore prepared has good biological activity and biocompatibility; Simultaneously, hyaluronic acid is connected by oxime key with amycin, it can discharge fast under tumor tissues or intracellular lower ph condition, thus enhancing drug effect, reduced form hyaluronic acid-the adriamycin bonding medicine with formula (II) structure also has more stable performance, more slow to the release of amycin, reach desirable long-acting treatment.
Description
Technical field
The present invention relates to chemical bonding medicine technical field, particularly relate to a kind of hyaluronic acid-adriamycin bonding medicine and preparation method thereof.
Background technology
Amycin, have another name called 1,4-Hydroxydaunomycin, Isosorbide-5-Nitrae-hydroxyl rubidomycin, many Suo Rou compare star, Doxorubicin, is a kind of antitumor drug of anthracycline antibiotic class high-efficiency broad spectrum, and is a kind of Cell cycle non-specific medicine and acts on the strongest to the S phase, mainly through inserting cell DNA, thus cause the tertiary structure of topoisomerase II destruction DNA and play drug effect.Up to now, amycin is considered to a kind of strong clinical chemotherapy medicine, the entity tumors such as primary treatment hepatocarcinoma, pulmonary carcinoma, gastric cancer, breast carcinoma, ovarian cancer, bladder cancer, thyroid carcinoma.
At present, the chemotherapy of amycin plays drug effect mainly through the mode of intravenously administrable clinically, but medicine distributes rapidly whole body after intravenous injection, in addition the amycin half-life is shorter, and the targets identification ability lacked tumor tissues, therefore tumor locus can be reached and to play the amycin ratio of curative effect very low, cause its bioavailability not high, inefficiency, clinically can only frequent drug administration in order to maintain drug effect, this causes larger toxic and side effects by the normal structure of health and organ, particularly heart, kidney.For solving the problem, the exploitation of amycin novel form is the focus of researcher research always, wherein, chemical bonding medicine obtains because drug encapsulation mode is more stable, drug release process is more lasting and pharmaceutical release time is more permanent and studies comparatively widely.
Publication number is that the Chinese patent of CN102406946A discloses a kind of polymer bond drug, first poly glycol monomethyl ether, carboxylated doxorubicin derivative, 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and N-hydroxy-succinamide are reacted in organic solvent, obtain reaction mixture, in reaction mixture, add PLL or chitosan again, after reaction, obtain high molecule adriamycin bonding medicine; Publication number is that the Chinese patent of 101234205A discloses a kind of high molecule adriamycin bonding medicine with target function, formed by two kinds of polyethylene glycol-polylactic acid block copolymer Hybrid assemblings, the polylactic acid end of the chain of the first polyethylene glycol-polylactic acid block copolymer is connected with amycin, the Polyethylene Glycol end of the chain of the second polyethylene glycol-polylactic acid block copolymer is connected with lactose, has slow-release function; Lactose has target function, can realize the targeted of amycin, therefore, this adriamycin bonding medicine can in tumor tissues slow release; But, above-mentioned two kinds of bonding medicines due to the chemical bond of bonding macromolecular compound and drug molecule too stable, thus there is the problems such as drug loading is low, the release lack of wisdom of amycin.Biomaterials(Vol.31, p1360-1371, 2010) a kind of carboxylated amycin that bonding poly glycol monomethyl ether and cis-3-carboxyl glutaconic anhydride are modified in the surface amino groups of polyamide-amide dendrimer is disclosed and the high molecule adriamycin bonding medicine that obtains, this bonding medicine can discharge fast under the acid condition of tumor tissues and cell, thus realize the intelligent of drug release, but the carrier material that this bonding medicine uses is polyamide-amide dendrimer, its preparation process is loaded down with trivial details, and poor biocompatibility, be unfavorable for the practical application of bonding medicine.
Summary of the invention
In view of this, the technical problem to be solved in the present invention is to provide a kind of hyaluronic acid-adriamycin bonding medicine and preparation method thereof, and the hyaluronic acid-adriamycin bonding medicine of preparation has good biocompatibility and pH response.
The invention provides a kind of hyaluronic acid-adriamycin bonding medicine, there is structure shown in formula (I) or formula (II):
Wherein, n is the hyaluronic degree of polymerization, 1≤n≤1290.
Present invention also offers a kind of preparation method of hyaluronic acid-adriamycin bonding medicine, comprising:
The buffer solution being dissolved with doxorubicin hydrochloride is mixed with hyaluronic acid, reacts, obtains the hyaluronic acid-adriamycin bonding medicine with formula (I) structure;
Or the buffer solution being dissolved with doxorubicin hydrochloride is mixed with hyaluronic acid and sodium cyanoborohydride, reacts, obtains the hyaluronic acid-adriamycin bonding medicine with formula (II) structure.
Preferably, described buffer solution is acetate buffer solution.
Preferably, the pH value of described buffer solution is 2 ~ 7.
Preferably, the temperature of described reaction is 20 DEG C ~ 65 DEG C.
Preferably, the time of described reaction is 12h ~ 120h.
Preferably, the mol ratio of described hyaluronic acid and amycin is 0.01 ~ 1; The mol ratio of described sodium cyanoborohydride and amycin is 0.01 ~ 1.
Preferably, also dialysis is comprised after described reaction.
Preferably, the pH value of described dialysis is 5 ~ 8.5.
Preferably, the time of described dialysis is 24h ~ 72h.
Compared with prior art, the invention provides a kind of hyaluronic acid-adriamycin bonding medicine, have structure shown in formula (I) or formula (II), wherein, n is the hyaluronic degree of polymerization, 1≤n≤1290.The present invention with hyaluronic acid, amycin for raw material, or with hyaluronic acid, sodium cyanoborohydride and amycin for raw material, two kinds of hyaluronic acid-adriamycin bonding medicines are prepared, because raw material hyaluronic acid has good biological activity and biocompatibility, the hyaluronic acid-amycin therefore prepared has good biological activity and biocompatibility; Simultaneously, hyaluronic acid is connected by oxime key with amycin, can discharge fast under its pH value condition lower in tumor tissues or cell, thus enhancing drug effect, reduced form hyaluronic acid-the adriamycin bonding medicine with formula (II) structure also has more stable performance, more slow to the release of amycin, reach desirable long-acting treatment.
Accompanying drawing explanation
Fig. 1 is the infrared absorption pattern of hyaluronic acid-adriamycin bonding medicine prepared by the embodiment of the present invention;
Fig. 2 is the hydrogen nuclear magnetic resonance spectrogram of hyaluronic acid-adriamycin bonding medicine prepared by the embodiment of the present invention;
Fig. 3 is the cumulative release curve chart of hyaluronic acid-adriamycin bonding medicine prepared by the embodiment of the present invention 2;
Fig. 4 is the cumulative release curve chart of hyaluronic acid-adriamycin bonding medicine prepared by the embodiment of the present invention 6.
Detailed description of the invention
The invention provides a kind of hyaluronic acid-adriamycin bonding medicine, there is structure shown in formula (I) or formula (II):
Wherein, n is the hyaluronic degree of polymerization, 1≤n≤1290.
The present invention with hyaluronic acid, amycin for raw material, or with hyaluronic acid, sodium cyanoborohydride and amycin for raw material, two kinds of hyaluronic acid-adriamycin bonding medicines are prepared, because raw material hyaluronic acid has good biological activity and biocompatibility, the hyaluronic acid-amycin therefore prepared has good biological activity and biocompatibility; Simultaneously, hyaluronic acid is connected by oxime key with amycin, can discharge fast under its pH value condition lower in tumor tissues or cell, thus enhancing drug effect, reduced form hyaluronic acid-the adriamycin bonding medicine with formula (II) structure also has more stable performance, more slow to the release of amycin, reach desirable long-acting treatment.
Hyaluronic acid-adriamycin bonding medicine provided by the invention has formula (I) or the structure shown in formula (II), and wherein, n is the hyaluronic degree of polymerization, preferably, 1≤n≤1290, preferred, 5≤n≤1000; The number-average molecular weight of described alginic acid is preferably 2000g.mol
-1~ 500000g.mol
-1, be more preferably 2500g.mol
-1~ 300000g.mol
-1, most preferably be 3000g.mol
-1~ 100000g.mol
-1; In the present invention, because the hyaluronic degree of polymerization of raw material used is controlled, therefore, the molecular weight of the hyaluronic acid-adriamycin bonding medicine of preparation is controlled.
Present invention also offers a kind of preparation method of hyaluronic acid-adriamycin bonding medicine, comprising:
The buffer solution being dissolved with doxorubicin hydrochloride is mixed with hyaluronic acid, reacts, obtains the hyaluronic acid-adriamycin bonding medicine with formula (I) structure;
Or the buffer solution being dissolved with doxorubicin hydrochloride is mixed with hyaluronic acid and sodium cyanoborohydride, reacts, obtains the hyaluronic acid-adriamycin bonding medicine with formula (II) structure.
Described hyaluronic acid-the adriamycin bonding medicine with formula (I) structure is prepared in accordance with the following methods:
First doxorubicin hydrochloride is dissolved in buffer solution and obtains mixed solution; Described doxorubicin hydrochloride has structure shown in formula III:
Described buffer solution is preferably acetate buffer solution, is specially sodium acetate-hac buffer; The pH value of described buffer solution is preferably 2 ~ 7, is more preferably 4.5 ~ 5.5, most preferably is 4.8 ~ 5.5; The ratio of the quality of described doxorubicin hydrochloride and the volume of buffer solution is preferably 0.01g ~ 1g:5mL.The present invention is preferred, and mix under the condition of lucifuge, the time of described mixing is preferably 10min ~ 60min, is more preferably 20min ~ 40min.
Then the mixed solution of preparation is mixed with hyaluronic acid, reacts, the hyaluronic acid-adriamycin bonding medicine with formula (I) structure can be prepared; Described hyaluronic acid has structure shown in formula IV:
Wherein, n is the hyaluronic degree of polymerization, 1≤n≤1290, and the mol ratio of described hyaluronic acid and amycin is preferably 0.01 ~ 1, is more preferably 0.05 ~ 1; Most preferably be 0.02 ~ 0.06; The temperature of described reaction is preferably 20 DEG C ~ 65 DEG C, is more preferably 30 DEG C ~ 60 DEG C, most preferably is 40 DEG C ~ 50 DEG C; The time of described reaction is preferably 12h ~ 120h, is more preferably 36h ~ 72h, most preferably is 48h ~ 60h; Described reaction is preferably carried out under the condition of lucifuge, and is preferably air-proof condition.
After reaction terminates, hyaluronic acid-the adriamycin bonding medicine with formula (I) structure of preparation is purified, the present invention is preferred, the method of dialysis is adopted to purify to hyaluronic acid-amycin, the method of the present invention to described dialysis there is no particular/special requirement, it can be dialysis process well known to those skilled in the art, the present invention preferably adopts bag filter to dialyse, described bag filter is preferably the bag filter that molecular cut off is 2483Dalton ~ 14000Dalton, be more preferably 7000Dalton, the pH value of described dialysis is preferably 5 ~ 8.5, be more preferably 6.0 ~ 8.0, most preferably be 6.5 ~ 7.8, the time of described dialysis is preferably 24h ~ 72h, is more preferably 36 ~ 72h, most preferably is 40h ~ 48h, concrete, first pH value adjustment is carried out to reaction system, preferably adopt sodium bicarbonate to regulate pH value of reaction system, then dialyse with bag filter, excessive free amycin dialysis is fallen, then filters and lyophilizing, hyaluronic acid-adriamycin bonding medicine sterling can be obtained.
The amino generation oximation reaction being hyaluronic aldehyde radical and doxorubicin hydrochloride at the described hyaluronic acid-adriamycin bonding medicine with formula (I) structure obtains.
Described hyaluronic acid-the adriamycin bonding medicine with formula (II) structure is prepared in accordance with the following methods:
First doxorubicin hydrochloride is dissolved in buffer solution and obtains mixed solution; Described buffer solution is preferably acetate buffer solution, is specially sodium acetate-hac buffer; The pH value of described buffer solution is preferably 2 ~ 7, is more preferably 4.5 ~ 5.5, most preferably is 4.8 ~ 5.5; The ratio of the quality of described doxorubicin hydrochloride and the volume of buffer solution is preferably 0.01g ~ 1g:5mL.
Then the mixed solution of preparation is mixed with hyaluronic acid and sodium cyanoborohydride, reacts, the hyaluronic acid-adriamycin bonding medicine with formula (II) structure can be prepared; Described sodium cyanoborohydride molecular formula is NaBH
3cN, its structure is such as formula shown in (V):
The mol ratio of described hyaluronic acid and amycin is preferably 0.01 ~ 1, is more preferably 0.05 ~ 1; The mol ratio of described sodium cyanoborohydride and amycin is preferably 0.01 ~ 1, is more preferably 0.05 ~ 0.5; The temperature of described reaction is preferably 20 DEG C ~ 65 DEG C, is more preferably 30 DEG C ~ 60 DEG C; The time of described reaction is preferably 12h ~ 120h, is more preferably 4h ~ 72h.
After reaction terminates, hyaluronic acid-the adriamycin bonding medicine with formula (II) structure of preparation is purified, the present invention is preferred, the method of dialysis is adopted to purify to hyaluronic acid-amycin, the method of the present invention to described dialysis there is no particular/special requirement, it can be dialysis process well known to those skilled in the art, the present invention preferably adopts bag filter to dialyse, described bag filter is preferably the bag filter that molecular cut off is 2483Dalton ~ 14000Dalton, the pH value of described dialysis is preferably 5 ~ 8.5, be more preferably 6.0 ~ 8.0, most preferably be 6.5 ~ 7.8, the time of described dialysis is preferably 24h ~ 72h, is more preferably 36h ~ 72h, most preferably is 40h ~ 48h, concrete, first pH value adjustment is carried out to reaction system, preferably adopt sodium bicarbonate to regulate pH value of reaction system, then dialyse with bag filter, excessive free amycin dialysis is fallen, then filters and lyophilizing, hyaluronic acid-adriamycin bonding medicine sterling can be obtained.
Have in the reduced form hyaluronic acid-adriamycin bonding medicine of formula (II) structure described, for the amino generation oximation reaction of hyaluronic aldehyde radical and amycin, and oxime key is reduced under the existence of sodium cyanoborohydride, obtain the reduced form hyaluronic acid-adriamycin bonding medicine with formula (II) structure.
The source of the present invention to described hyaluronic acid, sodium cyanoborohydride and doxorubicin hydrochloride there is no particular/special requirement, can be generally commercially available.
Carry out INFRARED ABSORPTION and magnetic resonance detection to the hyaluronic acid-adriamycin bonding medicine of preparation, result shows, in the present invention, together with hyaluronic acid and amycin are bonded to by oximation reaction, has prepared hyaluronic acid-adriamycin bonding medicine; Detect the drug release situation of hyaluronic acid-adriamycin bonding medicine under different pH condition of preparation, result shows, hyaluronic acid-adriamycin bonding medicine provided by the invention has good pH response.
The invention provides a kind of hyaluronic acid-adriamycin bonding medicine, have structure shown in formula (I) or formula (II), wherein, n is the hyaluronic degree of polymerization, 1≤n≤1290.The present invention with hyaluronic acid, amycin for raw material, or with hyaluronic acid, sodium cyanoborohydride and amycin for raw material, two kinds of hyaluronic acid-adriamycin bonding medicines are prepared, because raw material hyaluronic acid has good biological activity and biocompatibility, the hyaluronic acid-amycin therefore prepared has good biological activity and biocompatibility; Simultaneously, hyaluronic acid is connected by oxime key with amycin, can discharge fast under its pH value condition lower in tumor tissues or cell, thus enhancing drug effect, reduced form hyaluronic acid-the adriamycin bonding medicine with formula (II) structure also has more stable performance, more slow to the release of amycin, reach desirable long-acting treatment.
In order to further illustrate the present invention, below in conjunction with embodiment, hyaluronic acid-adriamycin bonding medicine provided by the invention and preparation method thereof is described in detail.
In following embodiment, product quality × 100% that reaction yield=actual product quality/theory obtained obtains.Described acetate buffer solution is specially sodium acetate-hac buffer.
Embodiment 1 ~ 8
The acetate buffer solution of preparation pH=5, the buffer respectively measuring 5mL configuration is placed in 8 round-bottomed flasks respectively, 0.05g doxorubicin hydrochloride is added respectively in each flask, after lucifuge stirring and dissolving, again according to the amount ratio of table 1, in each flask, add hyaluronic acid and sodium cyanoborohydride, described hyaluronic number-average molecular weight is 2483g.mol
-1, then each reaction bulb is sealed, according to the reaction condition of table 1, lucifuge reaction 52h, after reaction terminates, each reactant liquor pH to 7.4 is adjusted with sodium bicarbonate solution, then be the bag filter dialysis 48h of 7000Dalton with molecular cut off, lyophilizing after filtering, obtain hyaluronic acid-adriamycin bonding medicine respectively, experimental result is in table 1, table 1 is reaction condition and the productive rate of hyaluronic acid-adriamycin bonding medicine prepared by the embodiment of the present invention 1 ~ 8, wherein, i represents the mol ratio of hyaluronic acid and doxorubicin hydrochloride, j represents the mol ratio of sodium cyanoborohydride and doxorubicin hydrochloride.
INFRARED ABSORPTION detection and magnetic resonance detection are carried out to the hyaluronic acid-adriamycin bonding medicine of preparation, testing result is shown in Fig. 1 and Fig. 2, wherein, Fig. 1 is the infrared absorption pattern of hyaluronic acid-amycin prepared by the embodiment of the present invention, wherein, curve A is the infrared absorption pattern of hyaluronic acid-amycin prepared by embodiment 1, and curve B is the infrared absorption pattern of reduced form hyaluronic acid-amycin prepared by embodiment 5; Fig. 2 is the hydrogen nuclear magnetic resonance spectrogram of hyaluronic acid-amycin prepared by the embodiment of the present invention, wherein, curve A is the hydrogen nuclear magnetic resonance spectrogram of hyaluronic acid-amycin prepared by embodiment 1, and curve B is the hydrogen nuclear magnetic resonance spectrogram of reduced form hyaluronic acid-amycin prepared by embodiment 5; From Fig. 1 and Fig. 2, the present invention has prepared hyaluronic acid-adriamycin bonding medicine.
The reaction condition of hyaluronic acid-adriamycin bonding medicine prepared by table 1 embodiment of the present invention 1 ~ 8 and productive rate
| Embodiment | Temperature/DEG C | i | j | Reaction yield (%) |
| 1 | 30 | 0.1 | - | 73.4 |
| 2 | 40 | 0.1 | - | 92.2 |
| 3 | 50 | 0.1 | - | 82.0 |
| 4 | 60 | 0.1 | - | 72.6 |
| 5 | 30 | 0.1 | 0.3 | 81.8 |
| 6 | 40 | 0.1 | 0.3 | 95.8 |
| 7 | 50 | 0.1 | 0.3 | 82.1 |
| 8 | 60 | 0.1 | 0.3 | 81.0 |
Embodiment 9 ~ 16
The acetate buffer solution of preparation pH=5, the buffer respectively measuring 5mL configuration is placed in 8 round-bottomed flasks respectively, according to the proportioning of table 2, doxorubicin hydrochloride is added respectively in each flask, after lucifuge stirring and dissolving, again according to the amount ratio of table 2, in each flask, add hyaluronic acid and sodium cyanoborohydride, described hyaluronic number-average molecular weight is 2483g.mol
-1then each reaction bulb is sealed, 40 DEG C of lucifuge reaction 52h, after reaction terminates, each reactant liquor pH to 7.4 is adjusted with sodium bicarbonate solution, then be the bag filter dialysis 48h of 7000Dalton with molecular cut off, lyophilizing after filtering, obtain hyaluronic acid-adriamycin bonding medicine respectively, experimental result is in table 2, and table 2 is reaction condition and the productive rate of hyaluronic acid-adriamycin bonding medicine prepared by the embodiment of the present invention 9 ~ 16, wherein, i represents the mol ratio of hyaluronic acid and doxorubicin hydrochloride, and j represents the mol ratio of sodium cyanoborohydride and doxorubicin hydrochloride.
The reaction condition of hyaluronic acid-adriamycin bonding medicine prepared by table 2 embodiment of the present invention 9 ~ 16 and productive rate
| Embodiment | Doxorubicin hydrochloride consumption/g | i | j | Reaction yield (%) |
| 9 | 0.025 | 0.2 | - | 70.9 |
| 10 | 0.05 | 0.1 | - | 91.6 |
| 11 | 0.075 | 1:15 | - | 92.4 |
| 12 | 0.1 | 0.05 | - | 85.1 |
| 13 | 0.025 | 0.2 | 0.3 | 74.8 |
| 14 | 0.05 | 0.1 | 0.3 | 95.8 |
| 15 | 0.075 | 1:15 | 0.3 | 93.5 |
| 16 | 0.1 | 0.05 | 0.3 | 87.2 |
Embodiment 17 ~ 24
Acetate buffer solution is prepared respectively according to the pH data of table 3, and measure 5mL and be placed in round-bottomed flask, 0.05g doxorubicin hydrochloride is added respectively in each flask, after lucifuge stirring and dissolving, again according to the amount ratio of table 3, in each flask, add hyaluronic acid and sodium cyanoborohydride, described hyaluronic number-average molecular weight is 2483g.mol
-1then each reaction bulb is sealed, 40 DEG C of lucifuge reaction 52h, after reaction terminates, each reactant liquor pH to 7.4 is adjusted with sodium bicarbonate solution, then be the bag filter dialysis 48h of 7000Dalton with molecular cut off, lyophilizing after filtering, obtain hyaluronic acid-adriamycin bonding medicine respectively, experimental result is in table 3, and table 3 is reaction condition and the productive rate of hyaluronic acid-adriamycin bonding medicine prepared by the embodiment of the present invention 17 ~ 24, wherein, i represents the mol ratio of hyaluronic acid and doxorubicin hydrochloride, and j represents the mol ratio of sodium cyanoborohydride and doxorubicin hydrochloride.
The reaction condition of hyaluronic acid-adriamycin bonding medicine prepared by table 3 embodiment of the present invention 17 ~ 24 and productive rate
| Embodiment | pH | i | j | Reaction yield (%) |
| 17 | 4 | 0.1 | - | 92.5 |
| 18 | 4.5 | 0.1 | - | 94.4 |
| 19 | 5 | 0.1 | - | 93.5 |
| 20 | 5.5 | 0.1 | - | 92.2 |
| 21 | 4 | 0.1 | 0.3 | 93.5 |
| 22 | 4.5 | 0.1 | 0.3 | 93.7 |
| 23 | 5 | 0.1 | 0.3 | 95.8 |
| 24 | 5.5 | 0.1 | 0.3 | 92.6 |
Embodiment 25 ~ 32
The acetate buffer solution of preparation pH=5, the buffer respectively measuring 5mL configuration is placed in 8 round-bottomed flasks respectively, 0.05g doxorubicin hydrochloride is added respectively in each flask, after lucifuge stirring and dissolving, again according to the amount ratio of table 4, in each flask, add hyaluronic acid and sodium cyanoborohydride, described hyaluronic number-average molecular weight is 2483g.mol
-1, then each reaction bulb is sealed, at 40 DEG C, under the reaction condition of lucifuge, react according to the response time of table 4, after reaction terminates, each reactant liquor pH to 7.4 is adjusted with sodium bicarbonate solution, then be the bag filter dialysis 48h of 7000Dalton with molecular cut off, lyophilizing after filtering, obtain hyaluronic acid-adriamycin bonding medicine respectively, experimental result is in table 4, table 4 is reaction condition and the productive rate of hyaluronic acid-adriamycin bonding medicine prepared by the embodiment of the present invention 25 ~ 32, wherein, i represents the mol ratio of hyaluronic acid and doxorubicin hydrochloride, j represents the mol ratio of sodium cyanoborohydride and doxorubicin hydrochloride.
The reaction condition of hyaluronic acid-adriamycin bonding medicine prepared by table 4 embodiment of the present invention 25 ~ 32 and productive rate
| Embodiment | Response time | i | j | Reaction yield (%) |
| 25 | 32 | 0.1 | - | 80.3 |
| 26 | 42 | 0.1 | - | 84.5 |
| 27 | 52 | 0.1 | - | 92.6 |
| 28 | 62 | 0.1 | - | 91.0 |
| 29 | 32 | 0.1 | 0.3 | 78.1 |
| 30 | 42 | 0.1 | 0.3 | 83.6 |
| 31 | 52 | 0.1 | 0.3 | 95.8 |
| 32 | 62 | 0.1 | 0.3 | 94.8 |
Embodiment 33 ~ 40
The acetate buffer solution of preparation pH=5, the buffer respectively measuring 5mL configuration is placed in 8 round-bottomed flasks respectively, 0.05g doxorubicin hydrochloride is added respectively in each flask, after lucifuge stirring and dissolving, again according to the amount ratio of table 5, in each flask, add hyaluronic acid and sodium cyanoborohydride, described hyaluronic number-average molecular weight is 2483g.mol
-1, then each reaction bulb is sealed, 40 DEG C of lucifuge reaction 52h, after reaction terminates, according to the dialysis pH value data of table 5, regulate with the pH value of sodium bicarbonate solution to reactant liquor, then be the bag filter dialysis 48h of 7000Dalton with molecular cut off, lyophilizing after filtering, obtain hyaluronic acid-adriamycin bonding medicine respectively, experimental result is in table 5, table 5 is reaction condition and the productive rate of hyaluronic acid-adriamycin bonding medicine prepared by the embodiment of the present invention 33 ~ 40, wherein, i represents the mol ratio of hyaluronic acid and doxorubicin hydrochloride, j represents the mol ratio of sodium cyanoborohydride and doxorubicin hydrochloride.
The reaction condition of hyaluronic acid-adriamycin bonding medicine prepared by table 5 embodiment of the present invention 33 ~ 40 and productive rate
| Embodiment | Dialysis pH | i | j | Reaction yield (%) |
| 33 | 5.6 | 0.1 | - | 53.6 |
| 34 | 6.8 | 0.1 | - | 61.8 |
| 35 | 7.4 | 0.1 | - | 92.8 |
| 36 | 8.0 | 0.1 | - | 91.6 |
| 37 | 5.6 | 0.1 | 0.3 | 53.8 |
| 38 | 6.8 | 0.1 | 0.3 | 61.8 |
| 39 | 7.4 | 0.1 | 0.3 | 95.8 |
| 40 | 8.0 | 0.1 | 0.3 | 91.3 |
Embodiment 41 ~ 48
The acetate buffer solution of preparation pH=5, the buffer respectively measuring 5mL configuration is placed in 8 round-bottomed flasks respectively, 0.05g doxorubicin hydrochloride is added respectively in each flask, after lucifuge stirring and dissolving, again according to the amount ratio of table 6, in each flask, add hyaluronic acid and sodium cyanoborohydride, described hyaluronic number-average molecular weight is 2483g.mol
-1, then each reaction bulb is sealed, 40 DEG C of lucifuge reaction 52h, after reaction terminates, each reactant liquor pH to 7.4 is adjusted with sodium bicarbonate solution, then according to the dialysis time in table 6, dialyse with the bag filter that molecular cut off is 7000Dalton, lyophilizing after filtering, obtain hyaluronic acid-adriamycin bonding medicine respectively, experimental result is in table 6, table 6 is reaction condition and the productive rate of hyaluronic acid-adriamycin bonding medicine prepared by the embodiment of the present invention 41 ~ 48, wherein, i represents the mol ratio of hyaluronic acid and doxorubicin hydrochloride, j represents the mol ratio of sodium cyanoborohydride and doxorubicin hydrochloride.
The reaction condition of hyaluronic acid-adriamycin bonding medicine prepared by table 6 embodiment of the present invention 41 ~ 48 and productive rate
| Embodiment | Dialysis time (h) | i | j | Reaction yield (%) |
| 41 | 36 | 0.1 | - | —— |
| 42 | 48 | 0.1 | - | 91.2 |
| 43 | 60 | 0.1 | - | 82.8 |
| 44 | 72 | 0.1 | - | 72.4 |
| 45 | 36 | 0.1 | 0.3 | —— |
| 46 | 48 | 0.1 | 0.3 | 95.8 |
| 47 | 60 | 0.1 | 0.3 | 87.5 |
| 48 | 72 | 0.1 | 0.3 | 81.7 |
Wherein, embodiment 41 and 45, because dialysis time is too short, failing dialyses completely removes free amycin, and the hyaluronic acid-amycin purity of preparation is lower.
Embodiment 49 ~ 53
The acetate buffer solution of preparation pH=5, the buffer respectively measuring 5mL configuration is placed in 8 round-bottomed flasks respectively, 0.05g doxorubicin hydrochloride is added respectively in each flask, after lucifuge stirring and dissolving, again according to the amount ratio of table 7, in each flask, add hyaluronic acid and sodium cyanoborohydride, described hyaluronic number-average molecular weight is 2483g.mol
-1then each reaction bulb is sealed, 40 DEG C of lucifuge reaction 52h, after reaction terminates, each reactant liquor pH to 7.4 is adjusted with sodium bicarbonate solution, with the bag filter dialysis 48h that molecular cut off is 7000Dalton, lyophilizing after filtering, obtain hyaluronic acid-adriamycin bonding medicine respectively, experimental result is in table 7, and table 7 is reaction condition and the productive rate of hyaluronic acid-adriamycin bonding medicine prepared by the embodiment of the present invention 49 ~ 53, wherein, i represents the mol ratio of hyaluronic acid and doxorubicin hydrochloride, and j represents the mol ratio of sodium cyanoborohydride and doxorubicin hydrochloride.
Hyaluronic acid prepared by table 7 embodiment of the present invention 49 ~ 53
-the reaction condition of adriamycin bonding medicine and productive rate
| Embodiment | i | j | Reaction yield (%) |
| 49 | 0.1 | 1 | 79.8 |
| 50 | 0.1 | 0.3 | 95.8 |
| 51 | 0.1 | 0.2 | 93.3 |
| 52 | 0.1 | 0.1 | 91.6 |
| 53 | 0.1 | 1：15 | 89.9 |
Embodiment 54 ~ 63
The acetate buffer solution of preparation pH=5, the buffer respectively measuring 5mL configuration is placed in 8 round-bottomed flasks respectively, 0.05g doxorubicin hydrochloride is added respectively in each flask, after lucifuge stirring and dissolving, again according to the amount ratio of table 8, hyaluronic acid and sodium cyanoborohydride is added in each flask, described hyaluronic number-average molecular weight is in table 8, then each reaction bulb is sealed, 40 DEG C of lucifuge reaction 52h, after reaction terminates, each reactant liquor pH to 7.4 is adjusted with sodium bicarbonate solution, to dialyse 48h with bag filter, bag filter molecular cut off is in table 8, after having dialysed, filter and lyophilizing, obtain hyaluronic acid-adriamycin bonding medicine, experimental result is in table 8, table 8 is reaction condition and the productive rate of hyaluronic acid-adriamycin bonding medicine prepared by the embodiment of the present invention 54 ~ 63, wherein, i represents the mol ratio of hyaluronic acid and doxorubicin hydrochloride, j represents the mol ratio of sodium cyanoborohydride and doxorubicin hydrochloride.
Embodiment 54
Hyaluronic acid-adriamycin bonding medicine prepared by embodiment 2 and embodiment 6 is carried out drug release when pH value is 4.0,5.0,6.8,7.4 respectively, the results are shown in Figure 3 and Fig. 4, Fig. 3 is the cumulative release curve of hyaluronic acid-adriamycin bonding medicine prepared by the embodiment of the present invention 2, and Fig. 4 is the cumulative release curve of hyaluronic acid-adriamycin bonding medicine prepared by the embodiment of the present invention 6.
In Fig. 3, the percentage composition curve of the bonding medicine release amycin that hyaluronic acid-amycin that curve a, b, c, d are respectively embodiment 2 preparation records under pH value is respectively 4.0,5.0,6.8,7.4 conditions, pH is lower as seen from Figure 3, the release of amycin is faster, illustrate that this bonding medicine has good pH response, be bonded in amycin on hyaluronic acid can discharge fast under the pH condition of tumor tissues or tumor cell, thus strengthen effect of drugs.
In Fig. 4, for the percentage composition curve of the bonding medicine release amycin that the reduced form hyaluronic acid-amycin of embodiment 6 preparation records under pH value is respectively 4.0,5.0,6.8,7.4 conditions, pH change is very little on reduced form hyaluronic acid-adriamycin bonding medicine impact as seen from Figure 4, illustrate that it is more slow to the release of amycin, drug effect is longer.
From above-described embodiment and comparative example, the present invention for raw material, has prepared hyaluronic acid-adriamycin bonding medicine with hyaluronic acid, sodium cyanoborohydride and amycin, has good pH response.
The explanation of above embodiment just understands method of the present invention and core concept thereof for helping.It should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention, can also carry out some improvement and modification to the present invention, these improve and modify and also fall in the protection domain of the claims in the present invention.
Claims (10)
1. hyaluronic acid-adriamycin bonding medicine, has structure shown in formula (II):
Wherein, n is the hyaluronic degree of polymerization, 1≤n≤1290.
2. a preparation method for hyaluronic acid-adriamycin bonding medicine, comprising:
The buffer solution being dissolved with doxorubicin hydrochloride is mixed with hyaluronic acid and sodium cyanoborohydride, reacts, obtains the hyaluronic acid-adriamycin bonding medicine with formula (II) structure;
Wherein, n is the hyaluronic degree of polymerization, 1≤n≤1290.
3. method according to claim 2, is characterized in that, described buffer solution is acetate buffer solution.
4. method according to claim 2, is characterized in that, the pH value of described buffer solution is 2 ~ 7.
5. method according to claim 2, is characterized in that, the temperature of described reaction is 20 DEG C ~ 65 DEG C.
6. method according to claim 2, is characterized in that, the time of described reaction is 12h ~ 120h.
7. method according to claim 2, is characterized in that, the mol ratio of described hyaluronic acid and amycin is 0.01 ~ 1; The mol ratio of described sodium cyanoborohydride and amycin is 0.01 ~ 1.
8. method according to claim 2, is characterized in that, also comprises dialysis after described reaction.
9. method according to claim 8, is characterized in that, the pH value of described dialysis is 5 ~ 8.5.
10. method according to claim 8, is characterized in that, the time of described dialysis is 24h ~ 72h.
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